Increasing the Stability of DNA:RNA Duplexes by Introducing Stacking Phenyl-Substituted Pyrazole, Furan, and Triazole Moieties in the Major Groove

Article abstract of DOI:10.1021/acs.joc.5b01577

Consecutive incorporations of our previously published thymidine analogue, 5-(1-phenyl-1H-1,2,3-triazol-4-yl)-2′-deoxyuridine monomer W in oligonucleotides, has demonstrated significant duplex-stabilizing properties due to its efficient staking properties in the major groove of DNA:RNA duplexes. The corresponding 2′-deoxycytidine analogue is not as well-accommodated in duplexes, however, due to its clear preference for the ring-flipped coplanar conformation. In our present work, we have used ab initio calculations to design two new building blocks, 5-(5-phenylfuran-2-yl)-2′-deoxycytidine monomer Y and 5-(1-phenyl-1H-pyrazol-3-yl)-2′-deoxycytidine monomer Z, that emulate the conformation of W. These monomers were synthesized by Suzuki-Miyaura couplings, and the pyrazole moiety was obtained in a cycloaddition from N-phenylsydnone. We show that the novel analogues Y and Z engage in efficient stacking either with themselves or with W due to a better overlap of the aromatic moieties. Importantly, we demonstrate that this translates into very thermally stable DNA:RNA duplexes, thus making Y and especially Z good candidates for improving the binding affinities of oligonucleotide-based therapeutics. Since we now have both efficiently stacking T and C analogues in hand, any purine rich stretch can be effectively targeted using these simple analogues. Notably, we show that the introduction of the aromatic rings in the major groove does not significantly change the helical geometry.

Full text of DOI:10.1021/acs.joc.5b01577

Article
pubs.acs.org/joc
Increasing the Stability of DNA:RNA Duplexes by Introducing
Stacking Phenyl-Substituted Pyrazole, Furan, and Triazole Moieties
in the Major Groove
Mick Hornum, Pawan Kumar, Patricia Podsiadly, and Poul Nielsen*
Nucleic Acid Center, Department of Physics, Chemistry & Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230
Odense, Denmark
S
* Supporting Information
ABSTRACT: Consecutive incorporations of our previously published thymidine analogue, 5-(1-phenyl-1H-1,2,3-triazol-4-yl)-2′-
deoxyuridine monomer W in oligonucleotides, has demonstrated significant duplex-stabilizing properties due to its efficient
staking properties in the major groove of DNA:RNA duplexes. The corresponding 2′-deoxycytidine analogue is not as well-
accommodated in duplexes, however, due to its clear preference for the ring-flipped coplanar conformation. In our present work,
we have used ab initio calculations to design two new building blocks, 5-(5-phenylfuran-2-yl)-2′-deoxycytidine monomer Y and 5-
(1-phenyl-1H-pyrazol-3-yl)-2′-deoxycytidine monomer Z, that emulate the conformation of W. These monomers were
synthesized by Suzuki−Miyaura couplings, and the pyrazole moiety was obtained in a cycloaddition from N-phenylsydnone. We
show that the novel analogues Y and Z engage in efficient stacking either with themselves or with W due to a better overlap of
the aromatic moieties. Importantly, we demonstrate that this translates into very thermally stable DNA:RNA duplexes, thus
making Y and especially Z good candidates for improving the binding affinities of oligonucleotide-based therapeutics. Since we
now have both efficiently stacking T and C analogues in hand, any purine rich stretch can be effectively targeted using these
simple analogues. Notably, we show that the introduction of the aromatic rings in the major groove does not significantly change
the helical geometry.
improved, by the use of such analogues, they often lead to
structural and functional perturbation of the duplexes arising
from their non-natural sugar−phosphate backbone. For many
applications, however, it is important to maintain the native
secondary structure of the duplex in order to preserve the
biological activity. Less perturbing alternatives include mod-
ifications confined to the major groove faces of the nucleobases,
such as the 5-position of pyrimidines or the 7-position of
purines. In this way, the modified portion is placed discretely in
the major grooves of duplexes, thereby minimizing the structural
impacts on the duplex geometry as well as the Watson−Crick
base pairs.
INTRODUCTION
■
The high plasticity of oligonucleotides in combination with the
high specificity of Watson−Crick hybridization have made
oligonucleotides attractive agents for diagnostic1 and therapeu-
tic applications.2,3 Indeed, siRNA4 and antisense therapy5 are
promising alternatives to conventional chemotherapy of many
genetic disorders (including Duchenne muscular dystrophy,6
amyotrophic lateral sclerosis,7 etc.), cancers,8,9 viral infections,10
and other diseases for which the target mRNA can be identified.
However, for such oligonucleotides to become efficient
therapeutic agents, they need to be chemically modified to
optimize potency, biostability, absorption, and biodistribu-
tion.11,12 A number of nucleic acid analogues such as LNA13,14
have already provided oligonucleotides with significantly
increased hybridization affinity toward RNA; some of these
analogues are now widely used in many molecular biological
technologies,15 and some are entering clinics.16 Although the
hybridization characteristics are generally conserved, or even
It is well-known that favorable stacking of the heterocyclic
bases contributes most of the favorable enthalpy of duplex
formation for nucleic acid duplexes.17,18 Some efforts have been
Received: July 9, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
made to enhance π−π stacking in nucleic acids duplexes in order
to increase the thermal stabilities of the duplexes, including the
use of synthetic nucleobases with larger ring systems,19 such as
the tricyclic phenoxazine and phenothiazine nucleobase
analogues.20 More simple modifications have been to increase
the aromatic systems by connecting small (hetero)aromatic
moieties to the 5-position of pyrimidines.21,22 Along these lines,
we have previously shown that 1,2,3-triazoles,23 and in particular
1-phenyl-1H-1,2,3-triazol-4-yl moieties23−28 (monomers W and
X; Figure 1), in the 5-position of 2′-deoxyuridine and 2′-
to building block W in order to track down a simple C building
block with potentially optimized stacking interactions in the
major groove. After conformational analyses, we have designed
two new C building blocks: 5-(2-phenylfuran-5-yl)-2′-deoxy-
cytidine monomer Y and 5-(1-phenyl-1H-pyrazol-3-yl)-2′-
deoxycytidine monomer Z (Figure 1). First of all, we
demonstrate that these building blocks are well-accommodated
in DNA:RNA duplexes because of their anti positioning of the
phenyl substituent. This conformation places the phenyl group
into the hollow core of the major groove resembling monomer
W. Interestingly, we show that when W and Y and in particular
W and Z are placed adjacently in a 16-mer DNA:RNA duplex,
this geometrically optimized aromatic overlap translates into
significantly increased thermal stabilities of the duplexes relative
to the native ones without compromising the helical geometry.
RESULTS
■
Conformational Analyses. In continuation of our previous
conformational studies on phenyltriazoles,23,25,27 we decided to
use ab initio calculations to quantify the conformational
behaviors of potential five-membered aromatic heterocycles
directly attached to the 5-position of cytosine, in order to find
optimal candidates for a new C building block based on the
torsional profiles. Specifically, eight heterocycles were selected
and scanned for their tendency to form a torsional angle (θ) of
180° (anti) defined by the substituted face of the 5-heterocycle
and the C5,C6 face of cytosine. The choice of heterocycles for
the study was based on their likely capability of adopting an anti
conformation stabilized by an intermolecular O/N (hetero-
cycle)−HN (cytosine) hydrogen bond. All calculations were
performed at MP2/6-31G** in vacuum using simplified
derivatives of each compound.
In the case of all eight selected dimethylated heterocycle-
cytosine models, a clear preference for the anti conformation of
the C5−heterocycle bond was observed (see Figure S1 in
Supporting Information). Of the eight structures, 1-methyl-5-
(1-methyl-1H-pyrazol-3-yl)cytosine (representing monomer Z;
blue curve in Figure 2) showed a pronounced clear preference
for anti with a very deep global minimum at θ = 180° and with
the highest global maximum at θ = 0° of all the modeled
structures. The clear preference for the anti conformer suggests
that monomer Z is inclined to be geometrically similar to W
(black dashed line in Figure 2), which is essential for an efficient
aromatic overlap of the two residues. Similar energy profiles,
with a deep global minimum at θ = 180° and a global maximum
at θ = 0°, were obtained for three oxazole or thiazole cytosine
models (Figure S1, B-type profile).
Three other of the modeled structures, e.g., 1-methyl-5-(5-
methylfuran-2-yl)cytosine (representing monomer Y; red curve
in Figure 2), showed energy profiles with very wide minima at θ
= 180° (Figure S1, A-type profile). In these cases, the global
minima are in fact shifted slightly away from coplanarity by
about 30°; however, the low barrier separating the minima
(ΔE = 1−2 kJ/mol) means that they can essentially be treated as
a flat energy surface from 135° to 225°, indicating that they have
a high degree of rotational freedom in their anti conformations.
Although this less favorable energy profile does not directly
advocate efficient stacking, it nevertheless suggests that the
aromatic 5-substituents of these building block may be able to
adapt to their surrounding aromatic moieties, and in this way
potentially improve the π stacking interactions. Accordingly, we
decided to synthesize and incorporate both monomers Y and Z,
Figure 1. Structures of monomers W, X, Y, and Z.
deoxycytidine engage in efficient π−π stacking in the major
groove, which in turn translates into high-affinity RNA
recognition whenever two or more of these building blocks
are placed adjacently. In addition, oligonucleotides bearing these
building blocks show intact base pairing fidelity and high
stability toward nucleolytic degradation,25 making them
excellent candidates in siRNA or antisense designs.
Interestingly, we have shown that the DNA:RNA duplex
formation is significantly compromised if the linking triazole
group is either absent or even just inversed.27 Even more
delicately, we have shown that even the precise torsion of the
phenyltriazole moiety is crucial for optimal duplex stability, since
the subtle different orientations of the phenyltriazole moieties in
monomers W and X give rise to significant differences in the
thermal stability of the duplex. The preferred anti torsion of the
5-substituent of monomer W places the phenyl group toward
the core of the duplex, whereas the preferred syn torsion of
monomer X (Figure 1) points the phenyl group slightly away
from the helical axis.25 The anti torsion appears to be much
better accommodated by DNA:RNA duplexes, as evidenced by
comparing the changes in melting temperatures following single
(W, ΔTm = +1.8 °C; X, ΔTm = −2.5 °C) and multiple
(WWWW, ΔTm = +13.3 °C; XXXX, ΔTm = +4.9 °C)
incorporations of W and X centrally in a 16-mer DNA:RNA
duplex.25
In the present study, we have searched for alternative C
building blocks that are structurally and conformationally similar
B
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
a
Scheme 1
Figure 2. Energy scans of the torsion θ making up the single bond
between the two heterocycles of model compounds of W, X, Y, and Z
calculated using the MP2/6-31G** level. The illustrated structures are
drawn in their lowest-energy conformations.
the most appealing representatives of each energy profile types,
in duplexes to evaluate their stacking promise in vitro.
Synthesis. Oligonucleotides containing monomers W, Y,
and Z were synthesized on solid-phase using the natural
phosphoramidites along with the customized phosphoramidites
6 and 14 (Scheme 1) as well as the phosphoramidite of
monomer W. The phosphoramidite of W was synthesized from
5′-O-DMT-protected 5-ethynyl-2′-deoxyuridine in a CuAAC
reaction29,30 with azidobenzene, followed by a 3′-O-phosphity-
lation, using an improved protocol of our previous reported
procedure23 (see Supporting Information and Experimental
Section).
Phosphoramidite 6 was conveniently synthesized by a
Suzuki−Miyaura cross-coupling approach starting from the
totally deprotected 5-iodo-2′-deoxycytidine (1) and commercial
5-phenylfuran-2-boronic acid pinacol ester (2). This Pd-
catalyzed reaction was initially carried out in polar aprotic
solvents; however, in general, poor conversion of the starting
materials was observed under all reaction conditions, and
chromatographic separation from the residual starting material
was difficult. Realizing that the reaction progress was slightly
improved in water−organic cosolvent mixtures, we eventually
investigated the reactivity in pure water akin to Len and co-
workers.31 Using 10 mol % loading of the water-insoluble
catalyst, Pd(PPh3)4, and 2 equiv of NaOH, we remarkably
observed full conversion of the starting material 1 after just 60
min at 70 °C under microwave irradiation. A solid precipitated
from the water upon cooling of the reaction mixture, which,
when washed with a small amount of CH2Cl2 to remove the
catalyst, was found to be pure 5-(5-phenylfuran-2-yl)-2′-
deoxycytidine (3) in 75% yield. The reaction has been found
to be very robust, consistent, and scalable, and the fact that the
reaction proceeds in pure water solvent and without extractive
a
Reagents and conditions: (a) 2, Pd(PPh3)4, NaOH, H2O, 70 °C,
MW, 1 h, 75%; (b) N,N-dimethylformamide dimethylacetal, DMF, 55
°C, 2 h, 98%; (c) DMTCl, pyridine, rt, 16 h, 66% 5, 38% 13; (d)
P(N(i-Pr)2)O(CH2)2CN, diisopropylammonium tetrazolide, CH2Cl2,
rt, 18 h, 70% 6, 64% 14; (e) TBSCl, imidazole, DMF, rt, 16 h, 95%; (f)
9, 1,2-dichlorobenzene, 180 °C, MW, 4 h; then TBAF, THF, rt, 1 h,
29% (two steps); (g) 9, anisole, 165 °C, 36 h, 53%; (h) 12, Pd(PPh3)4,
K3PO4, DMF, H2O, 60 °C, 48 h, 80%. DMT = 4,4′-dimethoxytrityl.
or chromatographic workup makes this reaction a remarkably
convenient and green way to install a phenylfuran moiety to the
5-position of 2′-deoxycytidine. Nucleoside 3 was subsequently
protected with an amidine protecting group32 using N,N-
dimethylformamide dimethyl acetal in DMF in 98% yield,
followed by a 5′-O-selective tritylation using DMTCl in pyridine
to furnish the protected nucleoside 5 in 66% yield. Finally,
conversion into the corresponding 3′-O-phosphoramidite (6)
by treatment with 2-cyanoethyl N,N,N′,N′-tetraisopropylphos-
phorodiamidite (PN2) and diisopropylammonium tetrazolide in
CH2Cl2 proceeded in 70% yield.
For the synthesis of nucleoside 10 we investigated the use of a
thermal [2 + 3] sydnone−alkyne cycloaddition33 between 5-
ethynyl-2′-deoxycytidine34 (7) and N-phenylsydnone (9),35
C
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
which is a stable synthetic equivalent of the corresponding
azomethine imine, in order to construct the pyrazole ring at the
nucleoside level, akin to the alkyne−azide click reactions used
for the synthesis of the triazoles in W and X.23,25 Nucleoside 7
was protected to enhance the solubility in organic solvents and
to avoid nucleophilic opening of the mesoionic ring structure
under the high temperatures. In a series of attempts using
various temperatures (140−240 °C) and nonpolar solvents
(xylenes, toluene, 1,2-dichlorobenzene, anisole), it was found
that the cycloaddition proceeded optimally under microwave-
mediated heating to 180 °C in 1,2-dichlorobenzene and by the
use of transient tert-butyldimethylsilyl protecting groups for the
3′ and 5′ alcohols (compound 8), which were found to be
superior to acetyl protecting groups. Although we did not
manage to drive the cycloaddition to completion, 5-(1-phenyl-
1H-pyrazol-3-yl)-2′-deoxycytidine (10) was obtained in 29%
isolated yield following the subsequent desilylation. Also, 15% of
the corresponding 1,4-substituted pyrazole isomer was obtained,
but the two regioisomers could be separated by silica gel
chromatography. The two regioisomers were easily resolved on
boronate 12, the preparation and reactivity of which are known
in the literature.38 Accordingly, the sydnone−alkyne cyclo-
addition between commercial ethynylboronic acid MIDA ester
(11) and N-phenylsydnone (9) in anisole furnished compound
12, which was used for the subsequent Suzuki−Miyaura reaction
(Scheme 1). The MIDA boronate was found to be quickly
hydrolyzed and protodeboronated in aqueous NaOH solutions,
which severely hampered the reaction efficiency. However, with
the use of a K3PO4-mediated39 in situ slow release of the
unstable boronic acid, nucleoside 10 was obtained in 80% yield
from 5-iodo-2′-deoxycytidine (1) after 48 h at 60 °C in a
mixture of DMF and H2O (7:1) with 20 mol % of Pd(PPh3)4.
Nucleoside 10 was protected with a 5′-O-DMT group using
DMTCl in pyridine to obtain nucleoside 13 in 38% yield with
recovery of 52% unreacted nucleoside 10. The use of alternative
systems (Et3N, imidazole/CH2Cl2, 2,6-lutidine/DMSO,
DMAP/pyridine or AgNO3/pyridine) or higher temperatures
did not increase the isolated yield of nucleoside 13, in most
cases due to significant formation of the doubly or even triply
protected nucleoside products. Finally, nucleoside 13 was
phosphitylated at the 3′-position by PN2 to furnish the base-
unprotected phosphoramidite 14 in 70% yield. Puzzlingly, all
attempts to install acyl, carbamate, or amidine protecting groups
onto the nucleobase of 10 were unsuccessful, since the
protecting groups were found to be too labile, seemingly due
to their close proximity to the nucleophilic N2 (pyrazole)
position allowing for 6-endo-trig- or 6-exo-trig-type cyclizations,
followed by hydrolysis. This issue was not observed in the case
of nucleoside 3. We eventually decided to attempt oligonucleo-
tide synthesis with the base-unprotected phosphoramidite 14,
inspired by the published protocols for oligonucleotide synthesis
using phosphoramidites with unprotected bases.40−42
All phosphoramidites were successfully activated with 1H-
tetrazole in MeCN for the solid-phase oligonucleotide
syntheses. As expected, a significant amount of the N-
phosphitylated oligonucleotide products were formed whenever
phosphoramidite 14 was used. Although these defective
oligonucleotides could be removed from the desired oligonu-
cleotide later on by reversed-phase HPLC purification, we found
to our delight that replacing 1H-tetrazole with 1-hydroxybenzo-
triazole (HOBt) in MeCN-DMF (15:1, v/v) (inspired by Sekine
and co-workers42) provided 5′-O-specific condensation of the
upstream phosphoramidites without compromising coupling
yields. Accordingly, all three customized phosphoramidites were
efficiently incorporated into the 16-mer oligonucleotides listed
in Table 1 in excellent yields (>92%) using extended coupling
times (20 min). The structures and the purities of the
synthesized oligonucleotides were verified by MALDI-TOF
MS analysis and anion-exchange HPLC (see Supporting
Information).
1
the basis of the dissimilar H NMR coupling constants for the
two protons in their respective pyrazole rings: 3JHH4 = 2.5 Hz for
the 1,3-substituted pyrazole (nucleoside 10) and JHH ∼ 0−0.1
Hz for the 1,4-substituted pyrazole. Despite the recent
development of Cu-promoted sydnone−alkyne cycloaddi-
tions,36,37 no improvements in terms of reactivity or
regioselectivity were observed by the use of stoichiometric
amounts of Cu(OTf)2 or a few other Lewis acids.
To the best of our knowledge, we demonstrate here for the
first time the successful use of sydnone-alkyne cycloadditions for
the introduction of pyrazole rings into a highly functionalized
molecule. Although the isolated yield for the particular reaction
is rather low, this thermal cycloaddition strategy nevertheless
provides a feasible entry for pyrazole-functionalized nucleosides
from two simple starting materials (most of which could also be
recovered and recycled). In order to validate its potential on
other nucleoside substrates, we also attempted the cycloaddition
of N-phenylsydnone (9) with 5-ethynyl-2′-deoxyuridine (15) to
successfully form 5-(1-phenyl-1H-pyrazol-3-yl)-2′-deoxyuridine
(17) in 41% yield over three steps (Scheme 2). Also, small
amounts of the corresponding 1,4-substituted pyrazol isomer
were obtained.
In order to optimize yields for the synthesis of nucleoside 10,
we also investigated a Suzuki−Miyaura approach similar to the
synthesis of nucleoside 3 (Scheme 1). Due to the lack of a
commercial pyrazole equivalent of boronic acid pinacol ester 2,
we instead focused on the corresponding pyrazole MIDA
a
Scheme 2
Thermal Denaturation Studies. The synthesized oligonu-
cleotides listed in Table 1 were mixed in medium salt buffer
[Na2HPO4 (2.5 mM), NaH2PO4 (5 mM), NaCl (100 mM),
EDTA (0.1 mM), pH 7] with their complementary single-
stranded RNA and DNA sequences using 1.5 μM concen-
trations of each strand. Melting temperatures (Tm’s) of the
resulting duplexes (Table 1, entries 2−18 for DNA:RNA, and
Table S1, entries 2−18 for DNA:DNA) were derived from the
UV melting curves at 260 nm and compared with the native
duplex (Table 1/Table S1, entry 1).
a
As shown in Table 1, a single incorporation of C building
blocks Y and Z decreases the stability of the DNA:RNA
duplexes by −1.9 and −0.5 °C, respectively (entries 4 + 5),
(a) TBSCl, imidazole, DMF, rt, 4 h, quantitative; (b) 9, 1,2-
dichlorobenzene, 200 °C, MW, 5 h; (c) TBAF, THF, rt, 1 h, 41% (two
steps).
D
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
the 5′-WZ motif, possibly reflecting the different stacking
proportions of the aromatic moieties in the right-handed helix.
The Tm values of the DNA:DNA homoduplexes (Table S1)
featuring Y and Z show the overall same trend as their
corresponding DNA:RNA hybrid duplexes; however, the effect
of the stacking aromatic moieties appears less significant.
CD Spectroscopy. All synthesized DNA:RNA duplexes
were studied by CD spectroscopy in order to examine how the
modifications affect the overall conformation and secondary
structure of the duplexes. The CD spectra were recorded at 20
°C in medium salt buffer [Na2HPO4 (2.5 mM), NaH2PO4 (5
mM), NaCl (100 mM), EDTA (0.1 mM), pH 7] using 1.5 μM
concentrations of each strand. The obtained CD spectra of the
duplexes concerning Y are shown in Figure 3, and the spectra
concerning Z are shown in Figure 4. CD spectra of duplexes
featuring only X and W residues can be found in ref 25.
Table 1. Melting Temperatures of the Synthesized DNA:RNA
Duplexes
a
entry
ON sequences
Tm (ΔTm/mod) [°C]
b
1
5′-d(TTTTCTTTTCCCCCCT)
5′-d(TTTTCTTTWCCCCCCT)
5′-d(TTTTCTTTTXCCCCCT)
5′-d(TTTTCTTTTYCCCCCT)
5′-d(TTTTCTTTTZCCCCCT)
5′-d(TTTTCWWWWCCCCCCT)
5′-d(TTTTCTTTTXXXXCCT)
5′-d(TTTTCTTTTYYYYCCT)
5′-d(TTTTCTTTTZZZZCCT)
5′-d(TTTWXTTTTCCCCCCT)
5′-d(TTTWYTTTTCCCCCCT)
5′-d(TTTWZTTTTCCCCCCT)
5′-d(TTTTXWTTTCCCCCCT)
5′-d(TTTTYWTTTCCCCCCT)
5′-d(TTTTZWTTTCCCCCCT)
5′-d(TTTWXWTTTCCCCCCT)
5′-d(TTTWYWTTTCCCCCCT)
5′-d(TTTWZWTTTCCCCCCT)
62.7 or 60.0
b
2
64.5 (+1.8)
b
3
60.2 (−2.5)
4
58.1 (−1.9)
59.5 (−0.5)
5
b
6
76.0 (+3.3)
b
7
67.6 (+1.2)
8
66.7 (+1.7)
72.3 (+3.1)
9
b
10
11
12
13
14
15
16
17
18
68.7 (+3.0)
63.8 (+1.9)
64.9 (+2.5)
b
66.7 (+2.0)
63.1 (+1.6)
65.4 (+2.7)
b
70.9 (+2.7)
67.6 (+2.5)
70.2 (+3.4)
a
Melting temperatures (Tm’s) for DNA:RNA duplexes were obtained
from the maxima of the first derivatives of the absorbance (260 nm) vs
temperature curves. The samples contained 1.5 μM of each
oligonucleotide and 1.5 μM of complementary RNA 5′-r-
(GGGGGGAAAAGAAAA) in a phosphate buffered saline solution
(2.5 mM Na2HPO4, 5.0 mM NaH2PO4, 100 mM NaCl, 0.1 mM
EDTA, pH 7.0). Values in brackets show changes in the Tm values per
b
modification compared to the unmodified duplex. This value is taken
from ref 25 and is included in the table for comparison.
Figure 3. CD spectra of DNA:RNA duplexes featuring single (entry 4)
and multiple (entry 8) incorporations of monomer Y, as well as
duplexes containing consecutive W and Y building blocks (entry 11 +
14 + 17). The CD spectrum of the unmodified DNA:RNA duplex
(entry 1) is shown in black. Entry numbers refer to Table 1.
which are both less destabilizing than monomer X (ΔTm = −2.5
°C, entry 3), but more destabilizing than a single incorporation
of T building block W (ΔTm = +1.8 °C, entry 2) in this
sequence context. In turn, four consecutive incorporations of Y
and Z increase the melting temperature of the duplexes by +1.7
and +3.1 °C per modification (entries 8 + 9), which shows that
the thermal penalty of the incorporation appears to be more
than compensated by the improved π−π stacking of the
aromatic moieties. Importantly, the thermal stability of the
duplex featuring four Z residues practically reaches that of the
duplex featuring four W residues (ΔTm = +3.3 °C/mod, entry
6), which is remarkable considering the significant greater
change in the ΔTm upon going from 1 to 4 modifications [W,
ΔΔTm/mod = +1.5 °C (entry 2 vs 6); Z, ΔΔTm/mod = +3.6 °C
(entry 5 vs 9)].
As expected, the CD spectrum of the unmodified duplex
(black curve) adopts bands that are characteristic of an A/B-type
helix, including CD maxima near 270 nm (A-type) and 220 nm
(B-type), CD minima near 245 nm (B-type) and 210 nm (A-
type), and a clear crossover point (from positive to negative
intensity) at 250 nm, in agreement with a typical DNA:RNA CD
The three consecutive mixed stacking moieties WYW and
WZW increase the Tm’s by +2.5 and +3.4 °C per modification
(entries 17 + 18), respectively, relative to the unmodified
duplexes. This should be compared to the increase of +2.7 °C
per modification that was observed for the duplex featuring
WXW (entry 16). Thus, it appears that the expected better
aromatic overlap observed between the W and Z residues indeed
translates into a more thermally stable duplex in the case of the
WZW motif. On the other hand, the WYW motif is slightly less
stabilizing than WXW, which suggests that the rotationally less
restricted phenylfuran moiety causes less efficient stacking of the
aromatic 5-substituents. The same general tendency in the
thermal stability is seen for the DNA:RNA duplexes featuring
the 5′-XW, 5′-YW, and 5′-ZW motifs (entries 13−15). In the
case of the 5′-WX, WY, and WZ motifs (entries 10−12),
however, the 5′-WX motif is slightly better accommodated than
Figure 4. CD spectra of DNA:RNA duplexes featuring single (entry 5)
and multiple (entry 9) incorporations of monomer Z, as well as
duplexes containing consecutive W and Z building blocks (entry 12 +
15 + 18). The CD spectrum of the unmodified DNA:RNA duplex
(entry 1) is shown in black. Entry numbers refer to Table 1.
E
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 5. Global minimum structures obtained by 5 ns MD simulations of selected DNA:RNA duplexes from Table 1: (a) entry 1, (b) entry 16, (c)
entry 17, (d) entry 18, (e) entry 6, (f) entry 7, (g) entry 8, (h) entry 9. The calculations were performed at 300 K using the all-atom AMBER* force
field. The 5-substituents are shown with space-filling.
spectrum.43 As shown in both figures, the CD spectra of the
modified duplexes are strikingly similar to the CD spectrum of
the unmodified duplex, suggesting that the geometry changes
imposed by the modifications are indeed subtle. In fact, there
appears to be no general shift toward either an A- or B-type helix
in any of the duplexes. The most remarkable deviation from the
standard A/B-type profile is observed in the duplex featuring
four consecutive Z residues (Figure 4, entry 9), which contains a
significant positive band at 290 nm and a shoulder at 260 nm.
This new band appears to reflect local changes in the duplex
conformation due to stacking phenylpyrazole moieties.
Molecular Dynamics Simulations. To gain more
information about the geometries of the duplexes featuring
three and four consecutive modifications (Table 1, entries 6−9
and 16−18), a series of 5 ns molecular dynamics simulations
were performed. Using the all-atom AMBER* force field
(MacroModel V10.4, release 2014-2) on the structures in GB/
SA solvation, truthful global minimum structures of the
modified DNA:RNA duplexes in solution were obtained,
allowing us to visualize the degree of stacking of the 5-
substituents in the duplex as well as suggesting how the
modifications may influence the helical topology. The
minimized structures from the MD simulations are shown in
Figure 5. Top views are shown in Figure S2.
and especially W stack efficiently in the major groove (Figure
5e,f), and the present results show that also consecutive
incorporations of monomer Y and Z are able to stack very
efficiently (Figure 5g,h). As expected, the overall aromatic
overlaps in the WYW (Figure 5c) and WZW (Figure 5d) motifs
are much more efficient than the overlap in the WXW motif
(Figure 5b), due to the anti conformation of the 5-substituents
of the former two. However, in the case of the WYW motif, the
Y residue appears to be wobbling throughout the simulation (as
hinted by the ab initio calculations), and this may help explain
why the thermal stability of the duplex featuring the WYW motif
(Table 1, entry 17) is slightly reduced relative to that of the
WZW motif (Table 1, entry 18).
DISCUSSION
■
In this study, we have applied molecular modeling, CD
spectroscopy, and experimental melting temperatures to
investigate how heteroaromatic moieties attached to the 5-
position of pyrimidine nucleobases are accommodated in
DNA:RNA duplexes. Specifically, we have introduced two
new 5-substituted-2′-deoxycytidine monomers Y and Z, which
in turn place phenylfuran and phenylpyrazole moieties in the
major groove of the duplexes. These two monomers were
selected after ab initio studies of a pool of eight model
compounds due to their clear preferences for the anti torsion of
their 5-substituents, but with different rotational degrees of
freedom: the phenylfuran moiety of monomer Y is significantly
more free than the phenylpyrazole monomer Z. These two new
building blocks are synthesized in a few steps from commercially
available starting materials. For the synthesis of monomer Z, we
report the first use of a sydnone species reacting with a
As expected, the modeled DNA:RNA duplexes are all found
to be A/B-type helices with their 5-substitutents pointing into
the major grooves. In agreement with the CD spectra, the
modifications do not significantly alter the geometries of the
duplexes, except for a slight narrowing of the minor grooves at
the sites of the modifications. As previously shown by molecular
modeling,23,25 four consecutive incorporations of monomers X
F
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
nucleoside in a cycloaddition. This approach is strategically
similar to the azide−alkyne cycloaddition we have previously
used for the syntheses of monomers W and X.23,25 Also
remarkably, this study addresses the necessary but convenient
use of the N-unprotected phosphoramidite 14 as a substrate for
oligonucleotide synthesis. Successful 5′-O-selective condensa-
tion was achieved just by replacing the usual 1H-tetrazole
activator with HOBt, requiring no changes in the settings of the
synthesizer, and regular N-protected phosphoramidites were
used for the unmodified nucleotides without loss in coupling
yields.
shown by CD spectroscopy and molecular modeling. The highly
stabilizing effects observed for duplexes containing consecutive
W and Z residues appear to be due to a large aromatic overlap of
their 5-substituents. This makes the combination of the triazole-
functionalized thymidine analogue W and the pyrazole-
functionalized 2′-deoxycytidine analogue Z a simple tool in
high-affinity RNA-targeting oligonucleotides. In principle, a long
array of these stacking aromatic moieties can be used without
compromising the duplex geometry.
EXPERIMENTAL SECTION
■
From the hybridization studies, it is apparent that a single
incorporation of either monomer Y and Z leads to a small
decrease in the thermal stability of the duplex, possibly due to
distortion of the hydration of the major groove. However,
consecutive incorporations of either monomer Y and Z evoke
stabilizing effects due to favorable stacking of the aromatic
moieties, which more than compensates for the penalty of
placing the hydrophobic 5-substituents in contact with water.
The degree of stacking appears better in the case of monomer Z
than in the case of Y, suggesting that the phenylfuran moiety of
Y is too flexible for efficient π−π stacking interactions. This also
establishes a clear connection between the rotational profile and
the efficiency of stacking. Importantly, the stabilizing effects of
consecutive Z residues are noticeably superior to our previously
published C analogue X, and the stability gain is comparable to
our previously published T analogue W. The potential of
monomer Z is particularly clear when it is placed next to
monomer W, where an increased aromatic overlap between the
two similar-oriented 5-substituents appears to be the explan-
ation for a significantly increased Tm. In fact, the significant
stabilizing effect triggered by the WZW motif (ΔTm/mod =
+3.4 °C), roughly similar to those of WWWW (ΔTm/mod =
+3.3 °C) or ZZZZ (ΔTm/mod = +3.1 °C), signifies the
favorable π stacking interactions between these two T and C
analogues. Thus, monomer Z is well-suited as a complement to
monomer W in, e.g., RNA-targeting oligonucleotides for
obtaining high-affinity binding to complementary mixed purine
sequences. Notably, the Tm values of the sequence-identical
DNA:DNA duplexes are not nearly as stabilized by the stacking
aromatic moieties, indicative of preferential recognition of RNA
over DNA, possibly due to the more compressed character of
the A-type duplex over the B-type.
All reagents were used as supplied except CH2Cl2, which was distilled
prior to use. Microwave irradiated reactions were conducted in sealed
reaction vessels in a Biotage Initiator+ instrument using external surface
sensor probes. All reactions were monitored by TLC using silica gel (60
F254) precoated plates. Column chromatography was performed using
silica gel 60 (particle size 0.040−0.063 mm). For the purification of
DMT-protected nucleosides, the silica gel was pretreated with
pyridine/CH2Cl2 (1:100, v/v). After chromatography, appropriate
fractions were combined and concentrated. All products were dried in
high vacuum (<1 mm Hg) overnight to give products in high analytical
purity. HRMS-ESI was recorded on a quadrupole time-of-flight
1
instrument in positive ion mode with an accuracy of 5 ppm. H,
13C, and 31P NMR spectra were recorded at 400.12, 100.62, and 161.97
MHz, respectively. Chemical shifts are reported in ppm relative to
tetramethylsilane (δH,C 0 ppm) or the deuterated solvents (DMSO δc
39.5 ppm, CDCl3 δc 77.16 ppm). For 31P NMR spectra, 85% H3PO4
was used as external standard. 2D spectra (HSQC, COSY, and HMBC)
1
have been used in assigning H and 13C NMR signals.
5-(5-Phenylfuran-2-yl)-2′-deoxycytidine (3). 5-Iodo-2′-deoxy-
cytidine (1, 300 mg, 0.85 mmol) was dissolved in an aqueous solution
of NaOH (0.2 M, 4.25 mL, 0.85 mmol), and the solution was
completely degassed with N2. 5-Phenylfuran-2-ylboronic acid pinacol
ester (2, 275 mg, 1.02 mmol) and Pd(PPh3)4 (98 mg, 85 μmol) were
added, and the suspension was stirred under microwave irradiation for
60 min at 70 °C. The resulting yellow solution was cooled by N2 flow
for 15 min, and the resultant white precipitate was filtered off, washed
with cold CH2Cl2 (5 × 5 mL) and cold water (2 × 5 mL), and dried
under vacuum to obtain nucleoside 3 as a white solid (234 mg, 0.63
1
mmol). Yield: 75%. Rf 0.3 (6% MeOH in CH2Cl2). H NMR (400
MHz, DMSO-d6): δ 8.41 (s, 1H, H6), 7.76 (d, J = 7.5 Hz, 2H, H2 Ph),
7.73 (br, 1H, NH), 7.42 (t, J = 7.5 Hz, 2H, H3 Ph), 7.29 (t, J = 7.5 Hz,
1H, H4 Ph), 7.04 (d, J = 3.5 Hz, 1H, H4 furan), 6.75 (br, 1H, NH),
6.74 (d, J = 3.5 Hz, 1H, H3 furan), 6.22 (t, J = 6.4 Hz, 1H, H1′), 5.24
(d, J = 4.2 Hz, 1H, 3′-OH), 5.14 (t, J = 5.1 Hz, 1H, 5′-OH), 4.31−4.26
(m, 1H, H3′), 3.86 (dt, J = 3.2, 2.6 Hz, 1H, H4′), 3.71−3.59 (m, 2H,
H5′), 2.23 (ddd, J = 13.0, 6.4, 3.9 Hz, 1H, H2′), 2.11 (ddd, J = 13.0, 6.4,
6.0 Hz, 1H, H2′). 13C NMR (101 MHz, DMSO-d6): δ 161.6 (C4),
153.6 (C5 furan), 152.2 (C2), 146.9 (C2 furan), 139.9 (C6), 129.9 (C1
Ph), 128.7 (C3 Ph), 127.3 (C4 Ph), 123.4 (C2 Ph), 108.9 (C4 furan),
107.4 (C3 furan), 98.0 (C5), 87.5 (C4′), 85.5 (C1′), 70.0 (C3′), 61.0
(C5′), 40.9 (C2′). HRMS-ESI+: calcd for C19H19N3O5-H+ m/z
370.1397, found m/z 370.1397.
4-N-(N,N-Dimethylaminomethylidenyl)-5-(5-phenylfuran-2-
yl)-2′-deoxycytidine (4). To a solution of 5-(5-phenylfuran-2-yl)-2′-
deoxycytidine (3, 250 mg, 0.68 mmol) in anhydrous DMF (5 mL) was
added N,N-dimethylformamide dimethyl acetal (0.4 g, 3.4 mmol). The
reaction mixture was stirred for 2 h at 55 °C, and then concentrated
under reduced pressure. The residue was coevaporated with xylenes (2
× 5 mL), and the crude material was purified by flash chromatography
(0−10% MeOH in CH2Cl2) to obtain nucleoside 4 as a yellow solid
(282 mg, 0.66 mmol). Yield: 98%. Rf 0.4 (6% MeOH in CH2Cl2). 1H
NMR (400 MHz, DMSO-d6): δ 8.74 (s, 1H, H6), 8.72 (s, 1H, CH
amidine), 7.80 (d, J = 7.6 Hz, 2H, H2 Ph), 7.42 (t, J = 7.6, 2H, H3 Ph),
7.26 (t, J = 7.6 Hz, 1H, H4 Ph), 7.15 (d, J = 3.4 Hz, 1H, H4 furan), 6.99
(d, J = 3.4 Hz, 1H, H3 furan), 6.28 (t, J = 6.1 Hz, 1H, H1′), 5.30 (d, J =
4.2 Hz, 1H, 3′-OH), 5.22 (t, J = 5.3 Hz, 1H, 5′-OH), 4.33 (dt, J = 4.2,
3.4 Hz, 1H, H3′), 3.93 (q, J = 3.4 Hz, 1H, H4′), 3.75−3.69 (m, 2H,
As ascertained by CD spectroscopy and MD simulations, the
incorporation of the W, Y, and Z residues into the duplexes does
not significantly alter the geometries of the DNA:RNA duplexes
relative to the native DNA:RNA duplex, which is opposed to
many other known duplex stabilizing building blocks such as
LNA.44 Even so, the simple 5-modified pyrimidine building
blocks presented herein can also easily be used in combination
with other modifications such as phosphorothioates or LNA,
e.g., in therapeutic oligonucleotides.
CONCLUSION
■
In this study, we have investigated the directional influence of
phenyl-substituted heterocycles attached to the 5-position of
pyrimidines in perfecting the stacking of the aromatic moieties
in the major groove of DNA:RNA duplexes. Of the two new C
building blocks Y and Z presented herein, monomer Z
containing a phenylpyrazol moiety was found to engage in
highly favorable π−π stacking interactions in the major groove
with itself and with an adjacent T building block W. Notably, no
significant change in the duplex geometry was observed, as
G
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
H5′), 3.24 (s, 3H, CH3 amidine), 3.17 (s, 3H, CH3 amidine), 2.31
(ddd, J = 13.3, 6.1, 3.4 Hz, 1H, H2′), 2.17−2.09 (m, 1H, H2′). 13C
NMR (101 MHz, DMSO-d6): δ 166.1 (C2), 158.0 (C4), 153.6 (CH
amidine), 150.8 (C5 furan), 147.9 (C2 furan), 138.1 (C6), 130.2 (C1
Ph), 128.8 (C3 Ph), 127.1 (C4 Ph), 123.3 (C2 Ph), 110.8 (C4 furan),
107.6 (C3 furan), 104.9 (C5), 88.0 (C4′), 86.2 (C1′), 70.4 (C3′), 61.3
(C5′), 41.2 (C2′), 41.0 (CH3 amidine), 35.3 (CH3 amidine). HRMS-
ESI+: calcd for C22H24N4O5-H+ m/z 425.1819, found m/z 425.1808.
5′-O-(4,4′-Dimethoxytrityl)-4-N-(N,N-dimethylaminomethy-
lidenyl)-5-(5-phenylfuran-2-yl)-2′-deoxycytidine (5). To a sol-
ution of nucleoside 4 (280 mg, 0.66 mmol) in anhydrous pyridine (5
mL) was added DMTCl (400 mg, 1.0 mmol). The reaction mixture was
stirred for 16 h at rt. The reaction mixture was concentrated under
reduced pressure, coevaporated with toluene (2 × 5 mL), and purified
by flash chromatography (0−5% MeOH in CH2Cl2) to obtain
nucleoside 5 as a yellow foam (316 mg, 0.44 mmol). Yield: 66%. Rf
0.4 (4% MeOH in CH2Cl2). 1H NMR (400 MHz, CDCl3): δ 8.89 (s,
1H, H6), 8.34 (s, 1H, CH amidine), 7.44−7.36 (m, 4H, H2 Ph, DMT),
7.28−7.23 (m, 5H, H4 furan, DMT), 7.18−7.06 (m, 7H, H3 Ph, H4
Ph, DMT), 6.68−6.63 (m, 5H, DMT, H3 furan), 6.36 (t, J = 6.3 Hz,
1H, H1′), 4.33 (dt, J = 6.3, 4.0 Hz, 1H, H3′), 4.18 (q, J = 4.0 Hz, 1H,
H4′), 3.64 (s, 6H, OCH3), 3.52 (dd, J = 10.1, 4.0 Hz, 1H, H5′), 3.40
(dd, J = 10.1, 4.0 Hz, 1H, H5′), 3.22 (s, 3H, CH3 amidine), 3.21 (s, 3H,
(C5′), 42.6 (C2′), 26.1, 25.7 (SiC(CH3)3), 18.5, 18.0 (SiC(CH3)3),
−4.6, −4.9, −5.38, −5.41 (SiCH3). HRMS-ESI+: calcd for
C23H41N3O4Si2-Na+ m/z 502.2528, found m/z 502.2534.
N-Phenylsydnone (9). This material was synthesized by a known
method45 from N-phenylglycine. Mp 135.5−137 °C. H NMR (400
1
MHz, CDCl3): δ 7.76−7.72 (m, 2H, H3 Ph), 7.71−7.61 (m, 3H, H2
Ph, H4 Ph), 6.76 (s, 1H, H4 sydnone). 13C NMR (101 MHz, CDCl3):
δ 169.0 (C5 sydnone), 134.9 (C1 Ph), 132.5 (C4 Ph), 130.3 (C2 Ph),
121.3 (C3 Ph), 93.7 (C4 sydnone). IR (KBr, cm−1): v 3128 (sp2 CH
stretch), 1751 (CO stretch), 1472, 1440, 1176, 1087, 948, 854, 759,
726, 683. HRMS-ESI+: calcd for C8H6N2O2-Na+ m/z 185.0321, found
m/z 185.0316.
5-(1-Phenyl-1H-pyrazol-3-yl)-2′-deoxycytidine (10). For
method A, a sealed tube was charged with 3′,5′-bis-O-(tert-
butyldimethylsilyl)-5-ethynyl-2′-deoxycytidine (8, 53 mg, 0.11
mmol), N-phenylsydnone (9, 80 mg, 0.50 mmol), and 1,2-
dichlorobenzene (5 mL). The solution was stirred at 180 °C under
microwave irradiation for 4 h, upon which it was cooled, and the solvent
was removed under reduced pressure. The mixture of pyrazole products
was dissolved in THF (3 mL) and treated with a solution of TBAF in
THF (1 M, 0.25 mL, 0.25 mmol) for 1 h at rt. The solvent was
evaporated, and the two regioisomers were separated by flash
chromatography (0−15% MeOH in CH2Cl2). Nucleoside 10 was
obtained in the first fraction as a white solid (12 mg; 32 μmol). Yield:
29%. Rf 0.4 (10% MeOH in CH2Cl2). 1H NMR (400 MHz, DMSO-d6):
δ 8.71 (s, 1H, H6), 8.60 (d, J = 2.5 Hz, 1H, H5 pyrazole), 8.14 (br, 1H,
NH), 7.88 (br, 1H, NH), 7.87 (d, J = 7.7 Hz, 2H, H2 Ph), 7.52 (t, J =
7.7 Hz, 2H, H3 Ph), 7.32 (t, J = 7.7 Hz, 1H, H4 Ph), 6.84 (d, J = 2.5 Hz,
1H, H4 pyrazole), 6.21 (t, J = 6.2 Hz, 1H, H1′), 5.36 (t, J = 4.9 Hz, 1H,
5′-OH), 5.24 (d, J = 4.4 Hz, 1H, 3′-OH), 4.34−4.27 (m, 1H, H3′),
3.89−3.83 (m, 1H, H4′), 3.80−3.72 (m, 1H, H5′), 3.70−3.63 (m, 1H,
H5′), 2.24 (ddd, J = 4.6, 6.3, 13.2, 1H, H2′), 2.18−2.10 (m, 1H, H2′).
13C NMR (101 MHz, DMSO-d6): δ 162.1 (C4), 153.5 (C2), 148.2 (C3
pyrazole), 140.5 (C6), 139.0 (C1 Ph), 129.5 (C3 Ph), 128.9 (C5
pyrazole), 126.1 (C4 Ph), 118.0 (C2 Ph), 104.2 (C4 pyrazole), 98.3
(C5), 87.3 (C4′), 85.4 (C1′), 69.4 (C3′), 60.6 (C5′), 41.0 (C2′).
HRMS-ESI+: calcd for C18H19N5O4-H+ m/z 370.1510, found m/z
370.1512. The corresponding 1,4-substitued pyrazole isomer (5-(1-
phenyl-1H-pyrazol-4-yl)-2′-deoxycytidine) was obtained in the second
fraction as a white solid (6.0 mg; 16 μmol). Yield: 15%. Rf 0.35 (10%
CH3 amidine), 2.79−2.71 (m, 1H, H2′), 2.28−2.19 (m, 1H, H2′). 13
C
NMR (101 MHz, CDCl3): δ 167.2 (C2), 158.5 (DMT), 158.4 (C4),
154.9 (C5 furan), 152.2 (C2 furan), 148.0 (DMT), 144.6 (C6), 137.2
(CH amidine), 135.8, 135.5, 130.5, 130.0, 129.8, 128.6, 128.0, 127.9,
126.8, 123.5, 113.2 (DMT, Ph), 111.4 (C4 furan), 107.0 (C5), 106.5
(C3 furan), 87.3 (C1′), 86.7 (C4′), 85.9 (DMT), 72.6 (C3′), 63.8
(C5′), 55.1 (OCH3), 41.8 (C2′), 41.4 (CH3 amidine), 35.6 (CH3
amidine). HRMS-ESI+: calcd for C43H42N4O7-H+ m/z 727.3126, found
m/z 727.3129.
3′-O-(P-2-Cyanoethyl-N,N-diisopropylaminophosphinyl)-5′-
O-(4,4′-dimethoxytrityl)-4-N-(N,N-dimethylaminomethyliden-
yl)-5-(5-phenylfuran-2-yl)-2′-deoxycytidine (6). Nucleoside 5
(299 mg, 0.41 mmol) was coevaporated with anhydrous 1,2-
dichloroethane (2 × 3 mL) and dissolved in anhydrous CH2Cl2 (3
mL). Diisopropylammonium tetrazolide (139 mg, 0.82 mmol) and 2-
cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (248 mg, 0.82
mmol) were added, and the reaction mixture was stirred under argon
for 18 h at rt. The mixture was concentrated under reduced pressure,
and the residue was purified by flash chromatography (0−5% MeOH in
CH2Cl2) to afford a colorless oil. The oil was dissolved in CH2Cl2 (1
mL) and poured onto cold hexanes (100 mL). The resulting precipitate
was collected by decantation to obtain nucleoside phosphoramidite 6 as
a light yellow solid (268 mg, 0.29 mmol). Yield: 70%. Rf 0.35 (4%
MeOH in CH2Cl2). 31P NMR (162 MHz, CDCl3): δ 148.99, 148.50.
HRMS-ESI+: calcd for C52H59N6O8P-H+ m/z 927.4205, found m/z
927.4160.
3′,5′-Bis-O-(tert-butyldimethylsilyl)-5-ethynyl-2′-deoxycyti-
dine (8). 5-Ethynyl-2′-deoxycytidine (7, 0.60 g, 2.38 mmol) was
dissolved in anhydrous DMF (5 mL), and imidazole (0.73 g, 10.8
mmol) was added. The reaction mixture was stirred for 5 min, and
TBSCl (0.90 g, 5.97 mmol) was added in small portions. The reaction
mixture was stirred for 4 h, upon which saturated aqueous solution of
NaHCO3 (25 mL) was added. The mixture was extracted with CH2Cl2
(2 × 25 mL), and the combined organic extracts were washed with
water (50 mL), dried over MgSO4, and concentrated under reduced
pressure. The crude product was purified by flash column
chromatography (0−5% MeOH in CH2Cl2) to obtain nucleoside 11
as a white foam (1.14 g, 2.38 mmol). Yield: quantitative. Rf 0.4 (5%
MeOH in CH2Cl2). 1H NMR (400 MHz, CDCl3): δ 8.18 (s, 1H, H6),
7.36 (br, 1H, NH), 6.25 (t, J = 6.1 Hz, 1H, H1′), 5.73 (br, 1H, NH),
4.36 (dt, J = 6.1, 4.0 Hz, 1H, H3′), 3.97−3.90 (m, 2H, H4′, H5′), 3.77
(dd, J = 11.0, 2.0 Hz, 1H, H5′), 3.31 (s, 1H, CCH), 2.47 (ddd, J =
13.3, 6.1, 4.2 Hz, 1H, H2′), 2.03 (dt, J = 13.3, 6.1 Hz, 1H, H2′), 0.92 (s,
9H, SiC(CH3)3), 0.88 (s, 9H, SiC(CH3)3), 0.13 (s, 3H, SiCH3), 0.12 (s,
3H, SiCH3), 0.07 (s, 3H, SiCH3), 0.06 (s, 3H, SiCH3). 13C NMR (101
MHz, CDCl3): δ 164.7 (C4), 154.2 (C2), 144.9 (C6), 89.4 (CCH),
88.0 (C4′), 86.8 (C1′), 83.7 (CCH), 75.6 (C5), 71.4 (C3′), 62.4
1
MeOH in CH2Cl2). H NMR (400 MHz, DMSO-d6): δ 8.57 (s, 1H,
H6), 8.07 (s, 1H, H3 pyrazole), 7.88 (d, J = 8.5, 2H, Ph), 7.82 (s, 1H,
H5 pyrazole), 7.52 (t, J = 8.5, 2H, Ph), 7.32 (t, J = 8.5, 1H, Ph), 6.22 (t,
J = 6.5 Hz, 1H, H1′), 4.27 (dt, J = 7.3, 3.7 Hz, 1H, H3′), 3.79 (q, J = 3.5
Hz, 1H, H4′), 3.63 (dd, J = 11.8, 3.5 Hz, 1H, H5′), 3.59 (dd, J = 11.8,
3.5 Hz, 1H, H5′), 2.22−2.05 (m, 2H, H2′). 13C NMR (400 MHz,
DMSO-d6): δ 163.4 (C4), 154.3 (C2), 140.1 (C3 pyrazole), 139.52
(C3 pyrazole), 139.48 (C1 Ph), 129.4 (C3 Ph), 126.3 (C4 Ph), 126.2
(C6), 118.4 (C2 Ph), 115.7 (C4 pyrazole), 98.4 (C5), 87.2 (C4′), 85.0
(C1′), 69.8 (C3′), 60.8 (C5′), 40.6 (C2′). HRMS-ESI+: calcd for
C18H19N5O4-H+ m/z 370.1510, found m/z 370.1510. For method B, to
a degassed solution of 5-iodo-2′-deoxycytidine (1, 250 mg, 0.71 mmol)
and 1-phenyl-1H-pyrazol-3-boronic acid MIDA ester (12, 303 mg, 1.01
mmol) in DMF:H2O (7:1, 15 mL) were added K3PO4 (300 mg, 1.42
mmol) and Pd(PPh3)4 (164 mg, 0.14 mmol). The mixture was stirred
at 60 °C for 48 h, and then concentrated under reduced pressure and
purified by flash chromatography (0−10% MeOH in CH2Cl2) to obtain
nucleoside 9 as a white solid (210 mg; 0.57 mmol). Yield: 80%.
1-Phenyl-1H-pyrazol-3-boronic Acid MIDA Ester (12). N-
Phenylsydnone (9, 1.0 g, 6.17 mmol) and commercial ethynylboronic
acid MIDA ester (11, 1.34 g, 7.40 mmol) were suspended in anhydrous
anisole (25 mL) in a 50 mL steel bomb. The reaction chamber was
heated to 165 °C in an oven for 36 h, and slowly cooled to rt. The
reaction mixture was filtered, and the filtrate was concentrated under
reduced pressure; the two regioisomers were separated by flash
chromatography (50−100% EtOAc in hexanes). Compound 12 was
obtained in the first fraction as a light brown solid (0.98 g; 3.27 mmol).
1
Yield: 53%. Rf 0.5 (5% MeOH in CH2Cl2). H NMR (400 MHz,
DMSO-d6): δ 8.46 (d, J = 2.4 Hz, 1H, H5 pyrazole), 7.86 (d, J = 7.5 Hz,
2H, H2 Ph), 7.49 (t, J = 7.5 Hz, 2H, H3 Ph), 7.29 (t, J = 7.5 Hz, 1H, H4
H
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Ph), 6.58 (d, J = 2.4 Hz, 1H, H4 pyrazole), 4.36 (d, J = 17.1 Hz, 2H,
CH2), 4.11 (d, J = 17.1 Hz, 2H, CH2), 2.65 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6): δ 169.2 (CO), 139.8 (C1 Ph), 129.4 (C3
Ph), 127.7 (C5 pyrazole), 126.1 (C4 Ph), 118.6 (C2 Ph), 112.7 (C4
pyrazole), 104.4 (C3 pyrazole), 61.4 (CH2), 47.3 (CH3). HRMS-ESI+:
calcd for C14H14BN3O4-H+ m/z 300.1153, found m/z 300.1157. The
corresponding 1,4-substituted pyrazole isomer was obtained in the
second fraction as a light brown solid (0.34 g; 1.14 mmol). Yield: 19%.
(dd, J = 11.5, 2.5 Hz, 1H, H5′), 3.17 (s, 1H, CCH), 2.33 (ddd, J =
13.2, 5.9, 5.5 Hz, 1H, H2′), 2.03 (ddd, J = 13.2, 7.4, 5.5 Hz, 1H, H2′),
0.93 (s, 9H, SiC(CH3)3), 0.90 (s, 9H, SiC(CH3)3), 0.14 (s, 3H, SiCH3),
0.13 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3). 13C
NMR (101 MHz, CDCl3): δ 161.2 (C4), 149.0 (C2), 143.7 (C6), 99.0
(C5), 88.5 (C4′), 85.9 (C1′), 82.2 (CCH), 74.8 (CCH), 72.3
(C3′), 62.9 (C5′), 42.2 (C2′), 26.1, 25.7 (SiC(CH3)3), 18.5, 18.0
(SiC(CH3)3), −4.6, −4.8, −5.3, −5.4 (SiCH3). HRMS-ESI: calcd for
C23H40N2O5Si2-Na+ m/z 503.2368, found m/z 503.2352. To a solution
of 3′,5′-bis-O-(tert-butyldimethylsilyl)-5-ethynyl-2′-deoxyuridine (16,
670 mg, 1.39 mmol) in 1,2-dichlorobenzene (15 mL) was added N-
phenylsydnone (260 mg, 1.60 mmol). The mixture was stirred under
microwave irradiation for 5 h at 200 °C, and the black solution was
concentrated under reduced pressure. The residue was purified by flash
chromatography (0−80% EtOAc in hexanes) to obtain an inseparable
mixture of the two pyrazole isomers (about 4:1 ratio by 1H NMR) as a
yellow solid (441.2 mg, 0.74 mmol). Yield: 53%. Rf 0.65 (3% MeOH in
CH2Cl2). The mixture of two regioisomers (422 mg, 0.70 mmol) was
dissolved in THF (5 mL), and TBAF (1 M solution in THF; 0.77 mL,
0.77 mmol) was added. The reaction mixture was stirred for 1 h and
concentrated under reduced pressure. The residue was purified by flash
chromatography (0−6% MeOH in CH2Cl2). Nucleoside 17 was
obtained in the first fraction as a white solid (199 mg, 0.54 mmol).
1
Rf 0.45 (5% MeOH in CH2Cl2). H NMR (400 MHz, DMSO-d6): δ
8.40 (s, 1H, H5 pyrazole), 7.86 (d, J = 7.8 Hz, 2H, Ph), 7.71 (s, 1H, H3
pyrazole), 7.49 (t, J = 7.8 Hz, 2H, Ph), 7.29 (t, J = 7.8 Hz, 1H, Ph), 4.33
(d, J = 17.1 Hz, 2H, CH2), 4.11 (d, J = 17.1 Hz, 2H, CH2), 2.67 (s, 3H,
CH3). 13C NMR (400 MHz, DMSO-d6): δ 169.0 (CO), 144.8 (C3
pyrazole), 139.6 (C1 Ph), 131.4 (C5 pyrazole), 129.4 (C3 Ph), 125.9
(C4 Ph), 118.3 (C2 Ph), 99.4 (C4 pyrazole), 61.2 (CH2), 47.3 (CH3).
HRMS-ESI+: calcd for C14H14BN3O4-H+ m/z 300.1153, found m/z
300.1150.
5′-O-(4,4′-Dimethoxytrityl)-5-(1-phenyl-1H-pyrazol-3-yl)-2′-
deoxycytidine (13). To a solution of nucleoside 10 (326 mg; 0.88
mmol) in anhydrous pyridine (5 mL) was added DMTCl (419 mg;
1.24 mmol). The mixture was stirred at rt for 16 h, and then
concentrated under reduced pressure. The residue was purified by flash
chromatography (0−6% MeOH in CH2Cl2) to obtain unreacted
nucleoside 10 as a white solid (169 mg; 0.46 mmol) and nucleoside 13
as a light yellow foam (224 mg; 0.33 mmol). Yield: 38%. Rf 0.5 (5%
1
Yield: 77%. Rf 0.25 (5% MeOH in CH2Cl2). H NMR (400 MHz,
DMSO-d6, 17): δ 11.58 (br, 1H, NH), 8.60 (s, 1H, H6), 8.50 (d, J = 2.5
Hz, 1H, H5 pyrazole), 7.90 (dt, J = 8.5, 1.0 Hz, 2H, H2 Ph), 7.50 (ddd,
J = 8.5, 7.5, 2.0 Hz, 2H, H3 Ph), 7.30 (tt, J = 7.5 Hz, 1.0 Hz, H4 Ph),
6.96 (d, J = 2.5 Hz, 1H, H4 pyrazole), 6.25 (t, J = 6.7 Hz, 1H, H1′), 5.29
(d, J = 4.2 Hz, 1H, 3′-OH), 5.07 (t, J = 5.1 Hz, 1H, 5′−OH), 4.33−4.29
(m, 1H, H3′), 3.86 (q, J = 3.5 Hz, 1H, H4′), 3.65−3.62 (m, 2H, H5′),
2.23−2.19 (m, 2H, H2′). 13C NMR (101 MHz, DMSO-d6, 17): δ 161.5
(C4), 149.7 (C2), 145.5 (C3 pyrazole), 139.4 (C1 Ph), 137.1 (C6),
129.4 (C3 Ph), 128.0 (C4 Ph), 126.0 (C5 pyrazole), 118.1 (C2 Ph),
107.6 (C5), 106.6 (C4 pyrazole), 87.6 (C4′), 84.6 (C1′), 70.4 (C3′),
61.2 (C5′), 40.0 (C2′). The corresponding 1,4-substitued pyrazole
isomer (5-(1-phenyl-1H-pyrazol-4-yl)-2′-deoxyuridine) was obtained
in the second fraction as a white solid (18 mg, 0.05 mmol). Yield: 7%. Rf
1
MeOH in CH2Cl2). H NMR (400 MHz, CDCl3): δ 8.51 (br, 1H,
NH), 8.21 (s, 1H, H6), 7.57 (d, J = 7.6 Hz, 2H, H2 Ph), 7.54 (d, J = 2.5
Hz, 1H, H5 pyrazole), 7.48−7.39 (m, 4H, H3 Ph, DMT), 7.34−7.21
(m, 8H, DMT), 7.20−7.14 (m, 1H, H4 Ph), 6.77 (d, J = 8.0 Hz, 4H,
DMT), 6.61 (br, 1H, NH), 6.51 (t, J = 6.5 Hz, 1H, H1′), 5.87 (d, J = 2.5
Hz, 1H, H4 pyrazole), 4.54−4.47 (m, 1H, H3′), 4.29−4.21 (m, 1H,
H4′), 3.71 (s, 6H, DMT), 3.50 (dd, J = 10.4, 3.2 Hz, 1H, H5′), 3.30
(dd, J = 10.4, 4.0 Hz, 1H, H5′), 2.92−2.81 (m, 1H, H2′), 2.23 (dt, J =
13.3, 6.5 Hz, 1H, H2′). 13C NMR (101 MHz, CDCl3): δ 162.8 (C4),
158.6 (DMT), 155.0 (C2), 147.9 (DMT), 144.5 (C3 pyrazole), 139.5
(C1 Ph), 138.8 (C6), 135.7, 135.6, 130.1, 129.5, 128.2, 128.0 (DMT,
C3 Ph), 127.5 (C5 pyrazole), 127.0 (C4 Ph), 126.6 (DMT), 118.7 (C2
Ph), 113.2 (DMT), 104.2 (C4 pyrazole), 100.0 (C5), 86.8 (DMT),
86.6 (C4′), 86.2 (C1′), 71.9 (C3′), 63.7 (C5′), 55.19 (DMT), 42.3
(C2′). HRMS-ESI+: calcd for C39H37N5O6-Na+ m/z 694.2636, found
m/z 694.2617.
1
0.20 (5% MeOH in CH2Cl2). H NMR (400 MHz, CDCl3): δ 11.57
(br, 1H, NH), 8.68 (d, J = 0.1 Hz, 1H, H3 pyrazole), 8.47 (s, 1H, H6),
8.06 (d, J = 0.1 Hz, 1H, H5 pyrazole), 7.82 (dd, J = 8.5, 1.0 Hz, 2H, H2
Ph), 7.54−7.48 (m, 2H, H3 Ph), 7.32 (tt, J = 7.5, 1.0 Hz, 1H, H4 Ph),
6.23 (t, J = 6.7 Hz, 1H, H1′), 5.36 (t, J = 4.8 Hz, 1H, 5′-OH), 5.29 (d, J
= 4.3 Hz, 1H, 3′-OH), 4.36−4.31 (m, 1H, H3′), 3.84 (q, J = 3.3 Hz, 1H,
H4′), 3.73 (ddd, J = 11.8, 4.8, 3.3 Hz, 1H, H5′), 3.66 (ddd, J = 11.8, 4.4,
3.4 Hz, 1H, H5′), 2.27 (dt, J = 12.8, 6.3 Hz, 1H, H2′), 2.18 (ddd, J =
12.8, 6.7, 4.0 Hz, 1H, H2′). 13C NMR (101 MHz, DMSO-d6): δ 161.6
(C4), 149.5 (C2), 139.4 (C1 Ph), 138.4 (C5 pyrazole), 135.3 (C6),
129.5 (C3 Ph), 126.2 (C4 Ph), 124.4 (C3 pyrazole), 118.2 (C2 Ph),
116.1 (C4 pyrazole), 105.6 (C5), 87.4 (C4′), 84.4 (C1′), 69.7 (C3′),
60.7 (C5′), 39.9 (C2′). HRMS-ESI: calcd for C18H18N4O5-H+ m/z
371.1350, found m/z 371.1352.
3′-O-(P-2-Cyanoethyl-N,N-diisopropylaminophosphinyl)-5′-
O-(4,4′-dimethoxytrityl)-5-(1-phenyl-1H-1,2,3-triazol-4-yl)-2′-
deoxyuridine. To a solution of 5-ethynyl-2′-deoxyuridine (15, 498
mg, 1.97 mmol) in anhydrous pyridine (8 mL) was slowly added
DMTCl (0.80 g; 2.37 mmol). The reaction mixture was stirred for 16 h
at rt, quenched with MeOH (1 mL), and then concentrated under
reduced pressure. The crude material was coevaporated with toluene (2
× 10 mL), and purified by flash chromatography (0−5% MeOH in
CH2Cl2) to obtain 5′-O-(4,4′-dimethoxytrityl)-5-ethynyl-2′-deoxyur-
idine as a white foam (1.04 g; 1.88 mmol). Yield: 95%. Rf 0.35 (5%
MeOH in CH2Cl2). 1H NMR (400 MHz, DMSO-d6): δ 11.72 (br, 1H,
NH), 7.96 (s, 1H, H6), 6.10 (t, J = 6.7 Hz, 1H, H1′), 5.34 (d, J = 4.8
Hz, 1H, 3′-OH), 4.25 (td, J = 4.8, 3.7 Hz, 1H, H3′), 3.99 (s, 1H, C
CH), 3.93−3.89 (m, 1H, H4′), 3.74 (s, 6H, DMT), 3.24 (dd, J = 10.5,
5.4 Hz, 1H, H5′), 3.12 (dd, J = 10.5, 2.7 Hz, 1H, H5′), 2.30−2.24 (m,
1H, H2′), 2.19 (ddd, J = 13.5, 6.7, 3.7 Hz, 1H, H2′). 13C NMR (101
MHz, DMSO-d6): δ 161.6 (C4), 158.0 (DMT), 149.3 (C2), 144.8
(C6), 143.9, 135.5, 135.3, 129.7, 129.7, 127.9, 127.6, 126.7, 113.2
(DMT), 97.8 (C5), 85.8 (C4′), 85.0 (C1′), 83.7 (CCH), 75.8 (C
3′-O-(P-2-Cyanoethyl-N,N-diisopropylaminophosphinyl)-5′-
O-(4,4′-dimethoxytrityl)-5-(1-phenyl-1H-pyrazol-3-yl)-2′-deox-
ycytidine (14). Compound 13 (242 mg, 0.36 mmol) was
coevaporated with anhydrous 1,2-dichloroethane (2 × 3 mL) and
dissolved in anhydrous CH2Cl2 (2.5 mL). Diisopropylammonium
tetrazolide (122 mg, 0.72 mmol) and 2-cyanoethyl-N,N,N′,N′-
tetraisopropylphosphordiamidite (217 mg, 0.72 mmol) were added,
and the reaction mixture was stirred under argon for 18 h. The mixture
was concentrated under reduced pressure and purified by flash
chromatography (0−5% MeOH in CH2Cl2) to afford a colorless oil.
The oil was dissolved in CH2Cl2 (1 mL) and poured onto cold hexanes
(100 mL). The resulting precipitate was decanted and washed with cold
hexanes (50 mL) to obtain nucleoside phosphoramidite 14 as a white
solid (200 mg, 0.23 mmol). Yield: 64%. Rf 0.5 (3% MeOH in CH2Cl2).
31P NMR (162 MHz, CDCl3): δ 149.18, 148.47. HRMS-ESI+: calcd for
C48H54N7O7P−H+ m/z 872.3895, found m/z 872.3855.
5-(1-Phenyl-1H-pyrazol-3-yl)-2′-deoxyuridine (17). To a
stirred solution of 5-ethynyl-2′-deoxyuridine (15, 365 mg; 1.45
mmol) in anhydrous DMF (5 mL) was added imidazole (443 mg;
6.5 mmol). After 5 min, TBSCl (0.55 g; 3.62 mmol) was added in
portions, and the reaction mixture was stirred for 4 h at rt. A saturated
aqueous solution of NaHCO3 (25 mL) was added, and the mixture was
extracted with CH2Cl2 (2 × 25 mL). The combined organic phases
were washed with water (50 mL), dried over MgSO4, and concentrated
under reduced pressure to obtain nucleoside 16 as a white foam (695
mg; 1.45 mmol). Yield: quantitative. Rf 0.65 (5% MeOH in CH2Cl2).
1H NMR (400 MHz, CDCl3): δ 8.39 (br, 1H, NH), 8.10 (s, 1H, H6),
6.29 (dd, J = 7.4, 5.9 Hz, 1H, H1′), 4.41 (dt, J = 5.5, 2.5 Hz, 1H, H3′),
3.98 (q, J = 2.5 Hz, 1H, H4′), 3.91 (dd, J = 11.5, 2.5 Hz, 1H, H5′), 3.77
I
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
CH), 70.3 (C3′), 63.7 (C5′), 55.0 (DMT), 40.0 (C2′). HRMS-ESI:
calcd for C32H30N2O7-Na+ m/z 577.1945, found m/z 577.1949. A
microwave vial was charged with 5′-O-(4,4′-dimethoxytrityl)-5-ethynyl-
2′-deoxyuridine (450 mg, 0.81 mmol) and H2O/t-BuOH/pyridine
(2:2:1, 15 mL). Sodium ascorbate (96 mg, 0.49 mmol), CuSO4·5H2O
(40 mg, 0.17 mmol), and azidobenzene (3.25 mL, 1.62 mmol) were
added, and the reaction mixture was stirred for 1 h at 100 °C using
microwave irradiation. The reaction mixture was concentrated under
reduced pressure and purified by flash chromatography (0−10%
MeOH in CH2Cl2) to obtain 5′-O-(4,4′-dimethoxytrityl)-5-(1-phenyl-
1H-1,2,3-triazol-4-yl)-2′-deoxyuridine as a white foam (297 mg, 0.44
triethylammonium acetate (0.05 M, pH 7.4); eluent B = 75% MeCN/
H2O (3:1, v/v)). The pure fractions were pooled and evaporated at 45
°C. The 5′-terminal DMT group was removed by treatment with acetic
acid (80% in H2O, 100 μL) for 30 min, upon which an aqueous solution
of NaOAc (15 μL, 3 M), an aqueous solution of NaClO4 (15 μL, 5 M),
and pure acetone (1 mL) were added. The oligonucleotides
precipitated overnight at −20 °C. The supernatant was removed
from the sedimented solid (centrifugation, 12 000 rpm, 10 min at 2
°C), and the remaining pellet was washed with cold acetone (3 × 1 mL)
and dissolved in 500 μL of pure water. Analytically pure
oligonucleotides were obtained by preparative anion-exchange HPLC
(IE-HPLC) using a DIONEX Ultimate 3000 system equipped with
DNAPac PA100 semipreparative columns (13 μm, 250 mm × 9 mm)
heated to 60 °C. Elution was performed with an isocratic hold of 10%
eluent B, starting from 2 min hold on 2% eluent A in pure water,
followed by a linear gradient to 25% eluent A in 20 min at a flow rate of
2.0 mL/min (eluent A = NaClO4 (1.0 M); eluent B = TrisCl (0.25 M),
pH 8.0). The pure fractions were desalted using NAP-5 or NAP-10
Sephadex columns according to the manufacturer’s instructions, and
evaporated at 45 °C. Mass spectra of the oligonucleotides were
recorded on a MALDI-TOF MS instrument in ES+ mode. Analytical
IE-HPLC traces were recorded on a Merck-Hitachi Lachrom system
equipped with a DNAPac PA100 analytical column (13 μm, 250 mm ×
4 mm). The concentrations of the purified oligonucleotides were
determined by the optical density at 260 nm, assuming that the molar
absorptivities of the oligonucleotides equal the sum of each constituent
nucleotide monomers. The extinction coefficients of the modified
monomers in mL μmol−1 cm−1, ε260 (W) = 7.8, ε260 (Y) = 9.7, and ε260
(Z) = 14.6, were determined from the slopes of the absorbance of the
fully deprotected nucleosides at 25, 50, 75, and 100 μM concentrations
(R2 > 0.9995).
1
mmol). Yield: 73%. Rf 0.75 (5% MeOH in CH2Cl2). H NMR (400
MHz, DMSO-d6): δ 11.81 (s, 1H, NH), 8.82 (s, 1H, H6), 8.40 (s, 1H,
CH triazole), 7.91 (d, J = 7.5 Hz, 2H, H2 Ph), 7.60 (t, J = 7.5 Hz, 2H,
H3 Ph), 7.50 (t, J = 7.5 Hz, 1H, H4 Ph), 7.37 (d, J = 7.3 Hz, 2H, DMT),
7.31−7.20 (m, 4H, DMT), 7.20−7.10 (m, 3H, DMT), 6.82 (dd, J = 9.0,
2.0 Hz, 4H, DMT), 6.20 (t, J = 6.4 Hz, 1H, H1′), 5.36 (d, J = 6.8 Hz,
1H, 3′-OH), 4.21 (dt, J = 6.2, 4.2 Hz, 1H, H3′), 3.96 (q, J = 4.2 Hz, 1H,
H4′), 3.67 (s, 3H, DMT), 3.66 (s, 3H, DMT), 3.25−3.19 (m, 2H, H5′),
2.30−2.24 (m, 2H, H2′). 13C NMR (101 MHz, DMSO-d6): δ 161.1
(C4), 158.0 (C2), 149.6 (DMT), 144.8 (DMT), 139.8 (C1 Ph), 136.3
(C5 triazole), 135.5 (DMT), 135.4 (DMT), 129.9 (C3 Ph), 129.7
(DMT), 129.6 (C4 Ph), 127.8 (DMT), 127.6 (DMT), 126.6 (DMT),
120.1 (C2 Ph), 113.1 (DMT), 85.75 (C4′), 85.74 (C1′), 70.4 (C3′),
63.6 (C5′), 54.9 (DMT), 39.6 (C2′). HRMS-ESI: calcd for
C38H35N5O7-Na+ m/z 696.2429, found m/z 696.2417. 5′-O-(4,4′-
Dimethoxytrityl)-5-(1-phenyl-1H-1,2,3-triazol-4-yl)-2′-deoxyuridine
(205 mg, 0.30 mmol) was coevaporated with anhydrous 1,2-
dichloroethane (2 × 3 mL) and dissolved in anhydrous CH2Cl2 (3
mL). Diisopropylammonium tetrazolide (121.3 mg, 0.6 mmol) and 2-
cyano-N,N,N′,N′-tetraisopropylphosphordiamidite (183.4 mg; 0.6
mmol) were added, and the reaction mixture was stirred under argon
for 18 h. The mixture was concentrated under reduced pressure and
purified by flash chromatography (0−5% MeOH in CH2Cl2) to afford
crude material, which was dissolved in CH2Cl2 (1 mL) and poured onto
cold hexanes (100 mL). The resulting precipitate was collected by
decantation to obtain 3′-O-(P-2-cyanoethyl-N,N-diisopropylamino-
phosphinyl)-5′-O-(4,4′-dimethoxytrityl)-5-(1-phenyl-1H-1,2,3-triazol-
4-yl)-2′-deoxyuridine as a white solid (192.4 mg, 0.22 mmol). Yield:
73%. Rf 0.5 (5% MeOH in CH2Cl2). 31P NMR (162 MHz, CDCl3): δ
149.19, 148.74. HRMS-ESI: calcd for C47H52N7O8P-Na+ m/z
896.3507, found m/z 896.3478.
Melting Temperatures. The duplex samples consisted of 1.5 μM
concentrations of each oligonucleotide in medium salt buffer
[Na2HPO4 (2.5 mM), NaH2PO4 (5 mM), NaCl (100 mM), EDTA
(0.1 mM), pH 7]. The strands were annealed by heating the sample to
80 °C followed by a slow cooling to 10 °C. The increase in absorbance
at 260 nm as a function of temperature from 10 to 75 °C or 80 °C (1
°C/min) was recorded on a UV−vis spectrometer using a Peltier
Temperature Programmer. The listed Tm values are averages of at least
duplicate readings within 0.5 °C, as determined from the local
maximum of the first derivatives of the absorbance versus temperature
curves. All melting curves were found to be reversible.
Oligonucleotides. Oligonucleotides were synthesized on a fully
automated DNA synthesizer in ∼0.2 μmol scale loaded on 500 Å
controlled-pore glass (CPG) supports using the phosphoramidite
approach and following the manufacturer’s protocol. Double coupling
(2 × 5 min) cycles were used for commercial phosphoramidites, and
prolonged coupling times (20 min) were used for the modified
phosphoramidites 6 and 14. The phosphoramidites were activated
using 1H-tetrazole for 6 or HOBt for 14, and incorporated into
oligonucleotides via manual couplings: 10 μmol of the modified
phosphoramidite was dissolved in anhydrous MeCN (2 mL) and
treated with either 1H-tetrazole (3 mL, 0.45 M solution in MeCN) or
HOBt (3 mL, 0.3 M solution in 15:1 v/v MeCN/DMF) for 6 and 14,
respectively, and infused into the reaction compartment. The stepwise
coupling efficiencies were monitored by measuring the absorbance of
the trityl cation at 495 nm, which in all cases was 98−100% for the
commercial phosphoramidites, and 92−100% for the modified
phosphoramidites. The final 5′-terminal DMT group in the
oligonucleotides was kept on for purification purposes. The final
crude oligonucleotides on solid support were treated with NH3 (28% in
H2O, 1 mL) at 55 °C for 16 h. The mixture was filtered, and the filtrate
was evaporated to dryness at 45 °C by a steady N2 flow, and dissolved
in an aqueous triethylammonium acetate buffer (500 μL, 0.05 M, pH
7.4). The oligonucleotides were purified by reversed-phase HPLC on a
Waters 600 system using Xterra MS C18 10 μm (7.8 mm × 50 mm)
columns and Xterra MS C18 10 μm (7.8 mm × 10 mm) precolumns.
Elution was performed with 100% eluent A over 2 min, followed by a
linear gradient down to 30% eluent A over 38 min, then a wash with
100% eluent B over 10 min, 100% eluent A over 10 min (eluent A =
Circular Dichroism. CD spectra were recorded at 20 °C on a CD
spectrometer as an average of 5 scans from 200−350 nm using a split of
2.0 nm, and a scan speed of 50 nm/min. The samples were prepared
similarly to the samples used for the melting temperature studies with
1.5 μM concentrations of each strand. Quartz optical cells with a path
length of 5.0 mm were used.
Molecular Dynamics Simulations. The global minimum
structures were found from 5 ns molecular dynamics simulations
using the all-atom AMBER* force field applying the GB/SA solvation
model46 with extended cut-offs for nonbonded interactions (van der
Waals 8 Å and electrostatics 20 Å). Calculations were performed in
MacroModel V10.4 (within Maestro V9.8.017). The hybrid duplexes
were built in B-type helical geometries, and relaxed with AMBER*.
Initial Monte Carlo torsional samplings (MCMM) were performed to
generate 1000 structures, which were minimized into local minima. The
lowest energy structures of each simulations were used for the
subsequent MD simulations. The MD simulations were performed at
300 K. SHAKE all bonds to hydrogen was imposed in order to increase
the time step to 2.2 fs, and an equilibrium time of 100 ps was used to
stabilize the calculations. A multiple minimization of the 500 sampled
structures was performed to obtain a converged global minimum
structure.
Ab Initio Calculations. The torsional energy profiles were
calculated on the geometry optimized structures by a series of fully
relaxed coordinate scans of the dihedral angles between the nucleobases
and the 5-substituents in vacuum. The computations were performed in
Jaguar V7.8 (within Maestro V9.8.017) at the local MP2 level with the
6-31G** basis set, and by applying the Pipek−Mezey valence
J
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
localization method.47 The torsional sampling was performed by 2°
increments of the dihedral angles (from 0° to 360°) to obtain 181
structures, the relative energies of which were plotted against the
corresponding torsion angle.
(22) Ahmadian, M.; Bergstrom, D. E. In Modified Nucleosides;
Herdewijn, P., Ed.; Wiley-VCH Verlag GmbH & Co. KGaA: Berlin,
2008; pp 249−276.
(23) Koc
ChemBioChem 2007, 8, 2106−2116.
(24) Andersen, N. K.; Chandak, N.; Brulíkova,
̌
alka, P.; Andersen, N. K.; Jensen, F.; Nielsen, P.
́
L.; Kumar, P.; Jensen,
ASSOCIATED CONTENT
■
M. D.; Jensen, F.; Sharma, P. K.; Nielsen, P. Bioorg. Med. Chem. 2010,
18, 4702−4710.
S
* Supporting Information
(25) Andersen, N. K.; Døssing, H.; Jensen, F.; Vester, B.; Nielsen, P. J.
Org. Chem. 2011, 76, 6177−6187.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b01577.
(26) Kumar, P.; Chandak, N.; Nielsen, P.; Sharma, P. K. Bioorg. Med.
Chem. 2012, 20, 3843−3849.
Dihedral scan curves of the eight model compounds,
melting temperatures of DNA:DNA duplexes, optimized
synthesis of the phosphoramidite of monomer W,
selected NMR spectra, MALDI-TOF data of oligonucleo-
tides, analytical IE-HPLC profiles of oligonucleotides,
thermal denaturation curves, and top views of the
minimized duplex structures (PDF)
(27) Kumar, P.; Hornum, M.; Nielsen, L. J.; Enderlin, G.; Andersen,
N. K.; Len, C.; Herve,
2854−2863.
́
G.; Sartori, G.; Nielsen, P. J. Org. Chem. 2014, 79,
(28) Kaura, M.; Kumar, P.; Hrdlicka, P. J. Org. Biomol. Chem. 2012,
10, 8575−8578.
(29) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596−2599.
(30) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057−3064.
AUTHOR INFORMATION
■
(31) Gallagher-Duval, S.; Herve, G.; Sartori, G.; Enderlin, G.; Len, C.
New J. Chem. 2013, 37, 1989−1995.
Corresponding Author
(32) McBride, L. J.; Kierzek, R.; Beaucage, S. L.; Caruthers, M. H. J.
Am. Chem. Soc. 1986, 108, 2040−2048.
*E-mail: pouln@sdu.dk.
Notes
(33) Browne, D. L.; Harrity, J. P. A. Tetrahedron 2010, 66, 553−568.
(34) Barr, P. J.; Jones, A. S.; Serafinowski, P.; Walker, R. T. J. Chem.
Soc., Perkin Trans. 1 1978, 1263−1267.
The authors declare no competing financial interest.
(35) Baker, W.; Ollis, W. D. Nature 1946, 158, 703.
ACKNOWLEDGMENTS
■
́
(36) Comas-Barcelo, J.; Foster, R. S.; Fiser, B.; Gomez-Bengoa, E.;
The project was supported by The Danish Council for
Independent Research | Natural Sciences (FNU), and the
Villum Kann Rasmussen Fonden.
Harrity, J. P. A. Chem. - Eur. J. 2015, 21, 3257−3263.
(37) Kolodych, S.; Rasolofonjatovo, E.; Chaumontet, M.; Nevers, M.-
C.; Crem
12060.
́
inon, C.; Taran, F. Angew. Chem., Int. Ed. 2013, 52, 12056−
(38) Delaunay, T.; Es-Sayed, M.; Vors, J.-P.; Monteiro, N.; Balme, G.
Chem. Lett. 2011, 40, 1434−1436.
REFERENCES
■
(1) O’Connor, L.; Glynn, B. Expert Rev. Med. Devices 2010, 7, 529−
539.
(2) Kurreck, J. Therapeutic Oligonucleotides; Royal Society of
Chemistry: London, U.K., 2008.
(3) Goodchild. Methods Mol. Biol. 2011, 764, 1−15.
(4) Castanotto, D.; Rossi, J. J. Nature 2009, 457, 426−433.
(5) Dias, N.; Stein, C. A. Mol. Cancer Ther. 2002, 1, 347−355.
(6) Aartsma-Rus, A.; van Ommen, G.-J. B. RNA 2007, 13, 1609−
1624.
(7) Riboldi, G.; Zanetta, C.; Ranieri, M.; Nizzardo, M.; Simone, C.;
Magri, F.; Bresolin, N.; Comi, G. P.; Corti. Mol. Neurobiol. 2014, 50,
721−732.
(8) Gleave, M. E.; Monia, B. P. Nat. Rev. Cancer 2005, 5, 468−479.
(9) Devi, G. R. Cancer Gene Ther. 2006, 13, 819−829.
(10) Spurgers, K. B.; Sharkey, C. M.; Warfield, K. L.; Bavari, S.
Antiviral Res. 2008, 78, 26−36.
(11) Deleavey, G. F.; Damha, M. Chem. Biol. 2012, 19, 937−954.
(12) Watts, J. K.; Corey, D. R. J. Pathol. 2012, 226, 365−379.
(13) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 4, 455−456.
(39) Knapp, D. M.; Gillis, E. P.; Burke, M. D. J. Am. Chem. Soc. 2009,
131, 6961−6963.
(40) Gryaznov, S. M.; Letsinger, R. L. J. Am. Chem. Soc. 1991, 113,
5876−5877.
(41) Hayakawa, Y.; Kawai, R.; Kataoka, M. Eur. J. Pharm. Sci. 2001, 13,
5−16.
(42) Ohkubo, A.; Seio, K.; Sekine, M. Tetrahedron Lett. 2004, 45,
363−366.
(43) Bishop, G. R.; Chaires, J. B. Characterization of DNA Structures
by Circular Dichroism. Current Protocols in Nucleic Acid Chemistry;
Wiley: New York, 2003; Unit 7.11, pp 7.11.1−7.11.8.
(44) Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am.
Chem. Soc. 2002, 124, 5974−5982.
(45) Gribble, G. W.; Hirth, B. H. J. Heterocycl. Chem. 1996, 33, 719−
726.
(46) Qiu, D.; Shenkin, P. S.; Hollinger, F. P.; Still, W. C. J. Phys. Chem.
A 1997, 101, 3005−3014.
(47) Pipek, J.; Mezey, P. G. J. Chem. Phys. 1989, 90, 4916−4926.
(14) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.;
Imanishi, T. Tetrahedron Lett. 1998, 39, 5401−5404.
(15) Karkare, S.; Bhatnagar, D. Appl. Microbiol. Biotechnol. 2006, 71,
575−586.
(16) Zhang, L.-H.; Xi, Z.; Chattopadhyaya, J. Medicinal Chemistry of
Nucleic Acids; John Wiley & Sons: New York, 2011.
(17) Petersheim, M.; Turner, D. H. Biochemistry 1983, 22, 256−263.
(18) Yakovchuk, P.; Protozanova, E.; Frank-Kamenetskii, M. D.
Nucleic Acids Res. 2006, 34, 564−574.
(19) Luyten, I.; Herdewijn, P. Eur. J. Med. Chem. 1998, 33, 515−576.
(20) Lin, K.-Y.; Jones, R. J.; Matteucci, M. D. J. Am. Chem. Soc. 1995,
117, 3873−3874.
(21) Gutierrez, A. J.; Terhorst, T. J.; Matteucci, M. D.; Froehler, B. C.
J. Am. Chem. Soc. 1994, 116, 5540−5544.
K
DOI: 10.1021/acs.joc.5b01577
J. Org. Chem. XXXX, XXX, XXX−XXX